![]() Eas1 was expressed by fusing to a 6×His tag, and formed the NuA4 complex with the Epl1, Yng2, and Eaf6 subunits. (C) Co-expression of the NuA4 complex comprising four subunits. The complex was purified by glutathione affinity chromatography. Trim61 did not fuse to a tag but formed a complex with Trim6 fused to a GST tag. (B) Co-expression of the Trim6-Trim61 complex in the Trim6-Trim61-pSDB7 vector. The Trim6 protein fused to a 6×His tag was harvested using Ni-NTA resin, whereas the untagged Trim6 protein bound tightly with the Trim6 protein forming a complex, which was eluted with imidazole buffer. (A) Co-expression of the Trim6-Trim61 complex with two subunits of the Trim6-Trim61-pSDB1 vector. Thus, the HTFC technique is a simple, effective, reliable, and low-cost tool for parallel cloning. Furthermore, a method for generating polycistronic bacterial constructs based on the same strategy as that used for HTFC was developed. The target gene and vectors were PCR amplified separately to obtain the insert product and linear vectors with 18-base overlapping at each end of the DNAs required for FastCloning. Here, we describe a high-throughput FastCloning (HTFC) method, a protocol for parallel cloning by adding an adaptor sequence into all vectors. The situation could even be worse if multiple fragments of DNA are required to be added into one plasmid. ![]() However, parallel cloning of the gene into multiple vectors is still a labor-intensive operation, which requires a range of primers for different vectors in high-throughput cloning projects. Only two-step molecular manipulations are required to add a gene (cDNA) of interest into the desired vector. FastCloning, a reliable cloning technique for plasmid construction, is a widely used protocol in biomedical research laboratories. ![]()
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